Cholelithiasis in Thalassemia Major Patients: A Report from the South-East of Iran.

Background: Cholelithiasis and its predisposing factors are less characterized in thalassemia syndromes. In the present study, we assessed the prevalence of gallstones and related-risk factors among thalassemia major (TM) patients in south-east of Iran. Materials and Methods: The patients were recruited form a single center in Zabol city, south-east of Iran. Demographic and clinical information were retrieved from medical histories. Abdominal ultrasonography was performed to scrutinize gallstones and organ dimensions of liver, spleen, gallbladder and kidney. Results: The study participants (n=127) consisted of 50 (39.4%) males and 77 (60.6%) females. The mean age of the patients was 15.2±7.9 years. Cholelithiasis was observed in 11 (8.7%) patients. Cholelithiasis was significantly associated with age (P=0.002) and splenectomy (P=0.001). The patients with cholelithiasis received a significantly higher blood volume than patients without cholelithiasis (546±108.7 ml and 425.1±134.7 ml, respectively, P=0.007). There were significant differences between cholelithiasis and non- cholelithiasis TM patients regarding the length of right and left liver lobes (P=0.001), as well as the length of gallbladder (P=0.006). Ferritin level was not associated with cholelithiasis in our patients. In multivariate analysis, age older than 15 (OR=10.4, 95% CI: 1.2-86.3, P=0.02) and 30 years old (OR=42.6, 95% CI: 2.9-613, P=0.006), and splenectomy (OR=8.7, 95% CI: 2.1-35.4, P=0.002) were significant risk factors for cholelithiasis. Conclusion: Cholelithiasis is a relatively common complication among TM patients in our region. The most prominent risk factors of cholelithiasis were advanced age, splenectomy and large-volume blood transfusion.


INTRODUCTION
Thalassemia is the most common monogenic disorder and a global health issue. This syndrome is highly frequent in the Middle East countries including Iran. Thalassemia syndromes arise from deactivating mutations or deletions in alpha or beta globin genes 1,2 . Depending on the severity of the abrogation of globin chains output, thalassemia is classified into three main phenotypic categories:thalassemia minor, thalassemia intermediate (TI) and thalassemia major (TM) 3 . Among these clinically important forms, TI and TM are dependent on therapeutic interventions particularly regular transfusions. Although blood transfusions have resulted in magnificent improvement in the life expectancy of TM patients, secondary transfusion-related organ failure is a concerning issue among these patients.

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International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir Patients with TM need regular blood transfusions at two-to four-week intervals for ensuring sufficient tissue oxygenation. Transfusion -related hemosiderosis is considered as the main mechanism responsible for organ dysfunction in TM patients. The most common complications include cardiomyopathy, cholelithiasis, osteoporosis and endocrinopathies 4 . Despite notable achievements, current therapeutic protocols of TM are sufficient neither for prevention nor for management of these complications. Cholelithiasis is defined by the presence of radiological evidence of gallstones 5,6 . This complication has been reported in 10-57% of TM patients 5,[7][8][9][10] . Pathogenesis of cholelithiasis in TM is multifactorial. The main contributing factor is deemed to be precipitation of bilirubin in the bile as a result of increased hemolysis 11 .Iron deposition within gallbladder is also involved in the development of cholelithiasis 12 . The role of ineffective erythropoiesis has also been suggested in the formation of gallstones in TM 13,14 . Regarding inconsistencies over the pathogenesis and contributing factors in cholelithiasis, there is a need for further evaluation ofpotential mechanisms predisposing to cholelithiasis in TM. Sistan and Baluchistan province in the south-east of Iran is a region with high frequency of thalassemia in Iran 15,16 . Thalassemia patients in this province are unique regarding ethnic heterogenicity and genetic features. There were no previous studies on the prevalence and risk factors associated with cholelithiasis in TM in this region. In the present study, we evaluated 127 TM patients in Zabol city, the second most populated city in the province, regarding the prevalence of cholelithiasis and its risk factors.

MATERIALS AND METHODS
The current cross-sectional study was performed in Zabolcity,Sistan and Baluchestan province of Iran from April to September 2016. The study population included 127 TM patients in the only care center located in the city. The patients were under transfusion every 2-4 weeks. They were included into the study based on a targeted sampling method. The patients or their parents were requested to fill out an informed consent.
Demographic and clinical data were extracted frommedical records available at the center. Abdominal ultrasonography was performed for diagnosis of gallstones. Longitudinal and transverse planes were performed for seeking evidence of gallstones in hepatobiliary tree. Ultrasonography was also performed for determination of liver, spleenand kidney dimensions. Based on spleen length and upper limit of spleen size previously reported by Rosenberg et al. 17 , nonsplenectomized patients were categorized into groups of normal size, mild (<2 cm above upper limit) , moderate (2-4 cm above upper limit) and severe splenomegaly (>4 cm above upper limit) 9 . Statistical analysis was performed with SPSSVersion 19.0 statistic software package. Normality of data was assessed using Shapiro-Wilk test. Frequencies and descriptive statistics were used for presenting appropriate variables. Univariate analysis included Chi-square and independent-samples student's ttest. In multivariate analysis, logistic regression was used to predict risk factors for cholelithiasis.

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International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir Also, patients transfused with a higher mean blood volume showed higher ratio of cholelithiasis (Table  3).  the advanced age and splenectomy as the most prominent risk factors for cholelithiasis in our patients (Table 5).

DISCUSSION
Regarding the high rate of hemolysis and ineffective erythropoiesis, TM patients are susceptible to cholelithiasis, which can indicate thepresence of gallstones in gallbladder. In the current study, 116 TM patients from south-east of Iran were evaluated for evidence of cholelithiasis. For this purpose, the patients were undergone abdominal ultrasonography which is considered as a sensitive method for detecting cholelithiasis.
Overall, 11/127 (8.7%) patients were diagnosed with cholelithiasis. It was also reported in 29 (31.5%) among 92 TI patients in Shiraz, Iran 13 . In another study in North of Iran, cholelithiasis was reported in 17 (23.6%) out of 72 thalassemia patients 14 . Cholelithiasis was also described in 20.3% of TM and 57.1% of TI patients in Iran 10 .The prevalence of cholelithiasis in thalassemia carriers was reported as 20.3% 18 . Lotfi et al. evaluated 153 β-TM and 52 TI patients and reported cholelithiasis in 15 (9.8%) patients 9 . In a large population study on TI patients in Iran, 153 subjects were evaluated for thalassemia-related complications and cholelithiasis was reported in 25.5% of the participants 19 . This is while cholelithiasis has been reported in 0.1-0.3% of general pediatric population (20). This result was obtained while cholelithiasis was reported in 30-56% of TM patients in Italy 21,22 , 21.6% in the US 23 and 6-18% in Egypt 24,25 . Compared to previous studies, lower ratio of TM patients with cholelithiasis was identified in the present study. The relatively wide variation reported in different studies may reflect variations in patients' characteristics regarding demographic, transfusion history or genetic propensities 21,26,27 . Gilbert syndrome mutation has been the most dominant genetic contributor to formation of gallstones in thalassemia syndrome 18,28 . The rate of hemolysis also affects progression of cholelithiasis in TM. An elevated serum bilirubin level as a result of chronic hemolysis may also be involved in the formation of gallstones. The role of hemolysis, however, has been questioned by the fact that many TM patients with severe hemolysis do not form gallstones 22 . Despite the milder clinical severity and lower frequency of transfusions, the ratio of cholelithiasis has been higher in TI patients than TM 10 . This may highlight the potential role of disease-specific features in promoting gallbladder pathology in TM. The role of nutritional factors must be considered in propensity to cholelithiasis 29 . Previous studies indicated higher risk of gallstones in individuals consumingmoderate-high fat diets compared to those following low-fat diets 29 . Cholesterol as the major constituent accounts for most gallstones in patients with cholelithiasis 30 28 .
Higher pretransfusion hemoglobin level has also been noted as a risk factor for cholelithiasis in TM 25 . However, there was no significant difference between pretransfusion hemoglobin level of TM patients with and without cholelithiasis in our study. Although it has been suggested that female gender may be a risk factor for gallstones in general population 30 , we found no correlation between gender and cholelithiasis in our study which is in agreement with previous reports 9 . Totally, splenectomy, advanced age and large-volume blood transfusion seem to represent major risk factors for cholelithiasis in thalassemia patients. The role of these acquired parameters may be modified by genetic propensity of patients.

CONCLUSION
According to our results, gallstone is a relatively common complication in TM patients. The most significant predictors of cholelithiasis were advanced age, splenectomy and larger volume of blood transfusion. However, it seems that functional properties of gallbladder or genetic determinants may contribute to the risk of cholelithiasis. It is recommended to routinely evaluate TM patients, especially older patients, for the presence of cholelithiasis.